title: O-linked N-acetylglucosamine is present on the extracellular domain of notch receptors.
abstract: Rare types of glycosylation often occur in a domain-specific manner and are involved in specific biological processes. In particular, O-fucose glycans are reported to regulate the functions of EGF domain-containing proteins such as Notch receptors. In the course of mass spectrometric analysis of O-glycans displayed on Drosophila Notch receptors expressed in S2 cells, we found an unusual O-linked N-acetylhexosamine (HexNAc) modification which occurs at a site distinct from those of O-fucose and O-glucose glycosylations. Modification site mapping by mass spectrometry and amino acid substitution studies revealed that O-HexNAc modification occurs on a serine or threonine located between the fifth and sixth cysteines within the EGF domain. This modification occurs simultaneously along with other closely positioned O-glycosylations. This modification was determined to be O-beta-GlcNAc by galactosyltransferase labeling and beta-N-acetyl-hexosaminidase digestion experiments and by immunoblotting with a specific antibody. O-GlcNAc modification occurs at multiple sites on Notch epidermal growth factor repeats. O-GlcNAc modification was also found on the extracellular domain of Delta, a ligand for Notch receptors. Although the O-GlcNAc modification is known to regulate a wide range of cellular processes, the list of known modified proteins has previously been limited to intracellular proteins in animals. Thus, the finding of O-GlcNAc modification in extracellular environments predicts a distinct glycosylation process that might be associated with a novel regulatory mechanism for Notch receptor activity.
authors.affiliation: Department of Applied Molecular Biosciences, Nagoya University Graduate School of Bioagricultural Sciences, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Aiko
authors.initials: A
authors.lastname: Matsuura
authors.forename: Makiko
authors.initials: M
authors.lastname: Ito
authors.forename: Yuta
authors.initials: Y
authors.lastname: Sakaidani
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Kondo
authors.forename: Kosuke
authors.initials: K
authors.lastname: Murakami
authors.forename: Koichi
authors.initials: K
authors.lastname: Furukawa
authors.forename: Daita
authors.initials: D
authors.lastname: Nadano
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Matsuda
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Okajima
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D015870:Gene Expression; D006031:Glycosylation; D010449:Peptide Mapping / Q000379:methods; D046188:Protein Modification, Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000502:physiology; D051880:Receptors, Notch / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; C497759:N protein, Drosophila; D051880:Receptors, Notch; D000117:Acetylglucosamine